Broad Immune Monitoring and Profiling of T Cell Subsets with Mass Cytometry

Allergen immunotherapy can desensitize even subjects with potentially lethal allergies, but the changes induced in T cells that underpin successful immunotherapy remain poorly understood. In a cohort of peanut-allergic participants, we used allergen-specific T-cell sorting and single-cell gene expression to trace the transcriptional “roadmap” of individual CD4+ T cells throughout immunotherapy. We found that successful immunotherapy induces allergen-specific CD4+ T cells to expand and shift toward an “anergic” Th2 T-cell phenotype largely absent in both pretreatment participants and healthy controls. These findings show that sustained success, even after immunotherapy is withdrawn, is associated with the induction, expansion, and maintenance of immunotherapy-specific memory and naive T-cell phenotypes as early as 3 mo into immunotherapy. These results suggest an approach for immune monitoring participants undergoing immunotherapy to predict the success of future treatment and could have implications for immunotherapy targets in other diseases like cancer, autoimmune disease, and transplantation.

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Identification of Tr1 Cells and Other CD4+ T Cell Subsets in Humans Using Mass Cytometry: A Tool for Understanding Asthma

Journal of Allergy and Clinical Immunology ◽

10.1016/j.jaci.2015.12.1272 ◽

2016 ◽

Vol 137 (2) ◽

pp. AB410

Author(s):

Mary Prunicki ◽

Xiaoying Zhou ◽

Mariangels de Planell Saguer ◽

Rachel Miller ◽

Kari C. Nadeau

Keyword(s):

T Cell ◽

T Cell Subsets ◽

Cd4 T Cell ◽

Mass Cytometry ◽

Tr1 Cells

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Identification of Human Immune Cell Subtypes Most Vulnerable to IL-1β-induced Inflammatory Signaling Using Mass Cytometry

10.1101/2020.04.19.047274 ◽

2020 ◽

Author(s):

Hema Kothari ◽

Corey M. Williams ◽

Chantel McSkimming ◽

Mythili Vigneshwar ◽

Eli R. Zunder ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mononuclear Cells ◽

Immune Cell ◽

T Cell Subsets ◽

Effector Memory ◽

High Morbidity ◽

Cytokine Storm ◽

Mass Cytometry ◽

Immune Cell Subsets

ABSTRACTIL-1β has emerged as a key mediator of the cytokine storm linked to high morbidity and mortality from COVID-19 and blockade of the IL-1 receptor (IL-1R) with Anakinra has entered clinical trials in COVID-19 subjects. Yet, knowledge of the specific immune cell subsets targeted by IL-1β and IL-1β-induced signaling pathways in humans is limited. Utilizing mass cytometry (CyTOF) of human peripheral blood mononuclear cells, we identified effector memory CD4 T cells and CD4−CD8low/-CD161+ T cells as the circulating immune subtypes with the greatest expression of p-NF-κB in response to IL-1β stimulation. Notably, CCR6 distinctly identified T cells most responsive to IL-1β. Other subsets including CD11c myeloid dendritic cells (mDCs), classical monocytes (CM), two subsets of natural killer cells (CD16−CD56brightCD161− and CD16−CD56dimCD161+) and a population of lineage−(Lin-) cells expressing CD161 and CD25 also showed IL-1β-induced expression of p-NF-kB. The IL-1R antagonist, Anakinra significantly inhibited IL-1β-induced p-NF-kB in the CCR6+ T cells and CD11c mDCs with a trending inhibition in CD14 monocytes and Lin−CD161+CD25+ cells. IL-1β also induced a rapid but much less robust increase in p-p38 expression as compared to p-NF-kB in the majority of these same immune cell subsets. Prolonged IL-1β stimulation greatly increased p-STAT3 and to a much lesser extent p-STAT1 and p-STAT5 in T cell subsets, monocytes, DCs and the Lin−CD161+CD25+ cells suggesting IL-1β-induced production of downstream STAT-activating cytokines, consistent with its role in cytokine storm. Interindividual heterogeneity and inhibition of this activation by Anakinra raises the intriguing possibility that assays to measure IL-1β-induced p-NF-kB in CCR6+ T cell subtypes could identify those at higher risk of cytokine storm and those most likely to benefit from Anakinra therapy.

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FlowCT for the analysis of large immunophenotypic datasets and biomarker discovery in cancer immunology

Blood Advances ◽

10.1182/bloodadvances.2021005198 ◽

2021 ◽

Author(s):

Cirino Botta ◽

Catarina Da Silva Maia ◽

Juan-José Garcés ◽

Rosalinda Termini ◽

Cristina Perez ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Maintenance Therapy ◽

Large Scale ◽

Biomarker Discovery ◽

Immune Monitoring ◽

T Cell Subsets ◽

Multidimensional Data ◽

Modeling Tools ◽

Immune Biomarkers

Large-scale immune monitoring is becoming routinely used in clinical trials to identify determinants of treatment responsiveness, particularly to immunotherapies. Flow cytometry remains one of the most versatile and high throughput approaches for single-cell analysis; however, manual interpretation of multidimensional data poses a challenge to capture full cellular diversity and provide reproducible results. We present FlowCT, a semi-automated workspace empowered to analyze large datasets that includes pre-processing, normalization, multiple dimensionality reduction techniques, automated clustering and predictive modeling tools. As a proof of concept, we used FlowCT to compare the T cell compartment in bone marrow (BM) vs peripheral blood (PB) of patients with smoldering multiple myeloma (MM); identify minimally-invasive immune biomarkers of progression from smoldering to active MM; define prognostic T cell subsets in the BM of patients with active MM after treatment intensification; and assess the longitudinal effect of maintenance therapy in BM T cells. A total of 354 samples were analyzed and immune signatures predictive of malignant transformation in 150 smoldering MM patients (hazard ratio [HR]: 1.7; P <.001), and of progression-free (HR: 4.09; P <.0001) and overall survival (HR: 3.12; P =.047) in 100 active MM patients, were identified. New data also emerged about stem cell memory T cells, the concordance between immune profiles in BM vs PB and the immunomodulatory effect of maintenance therapy. FlowCT is a new open-source computational approach that can be readily implemented by research laboratories to perform quality-control, analyze high-dimensional data, unveil cellular diversity and objectively identify biomarkers in large immune monitoring studies.

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Mass cytometry analysis reveals a distinct immune environment in peritoneal fluid in endometriosis: a characterisation study

BMC Medicine ◽

10.1186/s12916-019-1470-y ◽

2020 ◽

Vol 18 (1) ◽

Cited By ~ 2

Author(s):

Manman Guo ◽

Cemsel Bafligil ◽

Thomas Tapmeier ◽

Carol Hubbard ◽

Sanjiv Manek ◽

...

Keyword(s):

T Cell ◽

Peritoneal Cavity ◽

Peritoneal Fluid ◽

Cell Function ◽

Immune Cell ◽

Adaptive Immune System ◽

T Cell Subsets ◽

Specific Cell ◽

Mass Cytometry ◽

Inflammatory Microenvironment

Abstract Background Endometriosis is a gynaecological condition characterised by immune cell infiltration and distinct inflammatory signatures found in the peritoneal cavity. In this study, we aim to characterise the immune microenvironment in samples isolated from the peritoneal cavity in patients with endometriosis. Methods We applied mass cytometry (CyTOF), a recently developed multiparameter single-cell technique, in order to characterise and quantify the immune cells found in peritoneal fluid and peripheral blood from endometriosis and control patients. Results Our results demonstrate the presence of more than 40 different distinct immune cell types within the peritoneal cavity. This suggests that there is a complex and highly heterogeneous inflammatory microenvironment underpinning the pathology of endometriosis. Stratification by clinical disease stages reveals a dynamic spectrum of cell signatures suggesting that adaptations in the inflammatory system occur due to the severity of the disease. Notably, among the inflammatory microenvironment in peritoneal fluid (PF), the presence of CD69+ T cell subsets is increased in endometriosis when compared to control patient samples. On these CD69+ cells, the expression of markers associated with T cell function are reduced in PF samples compared to blood. Comparisons between CD69+ and CD69− populations reveal distinct phenotypes across peritoneal T cell lineages. Taken together, our results suggest that both the innate and the adaptive immune system play roles in endometriosis. Conclusions This study provides a systematic characterisation of the specific immune environment in the peritoneal cavity and identifies cell immune signatures associated with endometriosis. Overall, our results provide novel insights into the specific cell phenotypes governing inflammation in patients with endometriosis. This prospective study offers a useful resource for understanding disease pathology and opportunities for identifying therapeutic targets.

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targetSCAPE and ultraSCAPE: Simultaneous identification and deep profiling of human antigen-specific T cells and other immune cell subsets by mass cytometry.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e15204 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e15204-e15204

Author(s):

Brian Abel ◽

Faris Kairi ◽

Alessandra Nardin ◽

Evan Newell ◽

Michael Fehlings

Keyword(s):

T Cells ◽

T Cell ◽

Immune Cell ◽

T Cell Subsets ◽

Single Sample ◽

High Dimensional ◽

Cell Populations ◽

Mass Cytometry ◽

Immune Cell Subsets

e15204 Background: During clinical trial immune monitoring, especially in the field of immunotherapy, it is critical to collect in-depth phenotypic information from multiple immune cell populations in order to assess the biological activity of the immunotherapy, to identify biomarkers of response or disease progression, and/or to identify new drug targets. However, patient samples such as peripheral blood mononuclear cells or tissues, are often scarce and current methods face limitations in either achieving a sufficient depth of analysis and/or cell throughput. Methods: In order to identify therapy-relevant antigens and to facilitate a concurrent in-depth characterization of T cells directed towards these targets, immunoSCAPE leverages the high-dimensional immune profiling capabilities of mass cytometry and a unique methodology allowing the identification and characterization of rare antigen-specific T-cell subsets (targetSCAPE). By implementing a new cutting-edge technology that combines flow and mass cytometry in parallel with a combinatorial live cell barcoding strategy, we further increased the high-dimensional phenotyping capacities to over 100 different marker molecules on up to four different immune cell subsets simultaneously within the same sample. Results: We isolated 4 different immune cell populations from a single sample and combined 3 different phenotypic panels consisting of 35 makers each together with a combinatorial tetramer multiplex and phenotyping panel for deep profiling of myeloid cells, NK cells, B cells and T cells. We demonstrate the potential of this novel immuno-phenotyping method, by tracking virus-specific T cells while simultaneously characterizing 4 immune cell subsets with over 100 distinct phenotypic markers from a single sample, which is currently impossible employing modern flow cytometers or classical mass cytometry methods. Conclusions: With its ability to provide an unprecedented picture of the immune status within a single sample, including T cell specificity information and in-depth profiling of relevant immune cell subsets, ultraSCAPE in combination with targetSCAPE can provide detailed insights on the effects of immunotherapy on the immune cell population. Information learned from in-depth immune phenotyping of several immune cell subsets such as T, B, NK and myeloid cell subsets can be leveraged for the development of novel diagnostics, biomarker discovery and monitoring therapeutic strategies in immunotherapy clinical trials.

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Are We Ready for the Use of Foxp3+Regulatory T Cells for Immunodiagnosis and Immunotherapy in Kidney Transplantation?

Journal of Transplantation ◽

10.1155/2012/397952 ◽

2012 ◽

Vol 2012 ◽

pp. 1-15 ◽

Cited By ~ 3

Author(s):

Francisco Salcido-Ochoa ◽

Nurhashikin Yusof ◽

Susan Swee-Shan Hue ◽

Doreen Haase ◽

Terence Kee ◽

...

Keyword(s):

T Cells ◽

Kidney Transplantation ◽

T Cell ◽

Regulatory T Cells ◽

Immune Monitoring ◽

T Cell Subsets ◽

Immunomodulatory Therapy ◽

Transplant Patients ◽

Phenotypic Characterisation ◽

Research Findings

The existence of T-cell subsets naturally committed to perform immunoregulation has led to enthusiastic efforts to investigate their role in the immunopathogenesis of transplantation. Being able to modulate alloresponses, regulatory T cells could be used as an immunodiagnostic tool in clinical kidney transplantation. Thus, the measurement of Foxp3 transcripts, the presence of regulatory T cells in kidney biopsies, and the phenotypic characterisation of the T-cell infiltrate could aid in the diagnosis of rejection and the immune monitoring and prediction of outcomes in kidney transplantation. Interestingly, the adoptive transfer of regulatory T cells in animal models has been proven to downmodulate powerful alloresponses, igniting translational research on their potential use as an immunomodulatory therapy. For busy transplant clinicians, the vast amount of information in the literature on regulatory T cells can be overwhelming. This paper aims to highlight the most applicable research findings on the use of regulatory T cells in the immune diagnosis and potential immunomodulatory therapy of kidney transplant patients. However, can we yet rely on differential regulatory T-cell profiles for the identification of rejection or to tailor patient's immunosuppression? Are we ready to administer regulatory T cells as inductive or adjunctive therapy for kidney transplantation?

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